Terminal differentiation within the erythroid lineage in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The interactions of erythroblasts with macrophages within the erythroblastic islands are critical for efficient enucleation. Defects in the final stages of erythropoiesis are common causes of anemia in chronic inflammatory diseases and neoplasia and in many primary hematological processes, like myelodysplasia and thalassemia. Limited understanding of the mechanisms involved in terminal erythroid maturation impedes development of novel therapies for such diseases. Using a novel analysis protocol of multiparameter high-speed cell imaging in flow we demonstrated evidence that enucleation is a multi-step process resembling asymmetric cytokinesis. It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocytes and pyrenocytes. Moreover, we showed that Rac GTPases organize actin in the actomyosin ring and aggregate lipid rafts in the furrow between nascent reticulocyte and pyrenocyte during enucleation. RhoA GTPase is known to play a significant role in cytokinesis and abscission of the daughter cells. Based on the resemblances between erythroblast enucleation and cytokinesis we hypothesize that Rac and RhoA dynamically control erythroblast enucleation by intrinsic molecular pathways analogous to the ones conducting cytokinesis. Based on the critical role of erythroblast-macrophage interaction for efficient erythropoiesis and the known function of Rac and RhoA in signaling initiated by integrins we hypothesize that Rac and RhoA transduce critical macrophage signals to the erythroblasts, regulating terminal erythroid differentiation and enucleation within the erythroblastic islands. To define the mechanistic contribution of RhoA signaling and the relationship of RhoA/Rac in regulating actin and microtubule cytoskeleton in the final stages of erythropoiesis, we bred mice with erythroid specific deletion (EpoRGFPcre/+ driven) of RhoA and Rac1/Rac2/Rac3. To test our hypotheses, we propose in Aim 1 to define the molecular processes by which RhoA/Rac-regulated cytoskeleton dynamics contribute to erythroblast enucleation and in Aim 2 to determine the role of Rho GTPases in enucleation and erythroblast maturation within erythroblastic islands. The goal of this proposal is to investigate the intracellular and intercellular molecular mechanisms by which Rac and RhoA regulate erythroblast differentiation and enucleation via dynamic regulation of actin and microtubule cytoskeletons. This study has the potential to reveal targets for in vivo therapeutic intervention for anemias due to terminal erythroid maturation defects as well as for improving the efficiency of red blood cell production in vitro. (End of Abstract)